Profiling of ERBB receptors and downstream pathways reveals selectivity and hidden properties of ERBB4 antagonists

Summary ERBB receptor tyrosine kinases are involved in development and diseases like cancer, cardiovascular, neurodevelopmental, and mental disorders. Although existing drugs target ERBB receptors, the next generation of drugs requires enhanced selectivity and understanding of physiological pathway responses to improve efficiency and reduce side effects. To address this, we developed a multilevel barcoded reporter profiling assay, termed ‘ERBBprofiler’, in living cells to monitor the activity of all ERBB targets and key physiological pathways simultaneously. This assay helps differentiate on-target therapeutic effects from off-target and off-pathway side effects of ERBB antagonists. To challenge the assay, eight established ERBB antagonists were profiled. Known effects were confirmed, and previously uncharacterized properties were discovered, such as pyrotinib’s preference for ERBB4 over EGFR. Additionally, two lead compounds selectively targeting ERBB4 were profiled, showing promise for clinical trials. Taken together, this multiparametric profiling approach can guide early-stage drug development and lead to improved future therapeutic interventions.


INTRODUCTION
2][3][4] The ERBB family consists of four type I transmembrane receptors, which are the epidermal growth factor receptor (EGFR, also known as ERBB1), ERBB2 receptor tyrosine kinase 2 (also known as HER2), ERBB3 (HER3), and ERBB4 (HER4). 2 ERBB receptors are activated by epidermal growth factor (EGF) and heregulin-like family ligands and can homo-and heterodimerize, leading to the phosphorylation on cytoplasmic tyrosine residues and the activation of cellular signaling cascades.6][7] ERBB3 favors heterodimerization with its preferred dimerization partner ERBB2 due to an impaired kinase domain to elicit cellular signaling. 8Canonical cellular signaling pathways initiated by activated ERBB receptors are the KRAS-RAFmitogen-activated protein kinase (MAPK) cascade, phosphatidylinositol 3-kinase (PI3-K), and STAT signaling. 9,10In particular, the MAPK axis, which is initiated by the binding of the growth factor receptor-bound protein 2 (GRB2) adapter to phosphorylated tyrosine residues of ERBB receptors and ultimately results in extracellular regulated kinase 1 and 2 (ERK1/2) activation, is a bona-fide pathway that links ERBB receptor activity to cellular phenotypes, like cell cycle progression and proliferation. 11,12ancer formation is linked to an increased expression of EGFR, 13 ERBB2, 14 ERBB3, 15 and ERBB4. 16,17Notably, ERBB receptors play overlapping roles in various cancers, with, however, distinct mechanisms. 18For example, both EGFR and ERBB4 are linked to colorectal cancer 19,20 and non-small cell lung cancer (NSCLC), 21 but mutations for either EGFR or ERBB4 generally occur separately.Inhibiting ERBB signaling using small molecules is an attractive approach to treat multiple types of cancer.For example, gefitinib, the first EGFR tyrosine kinase inhibitor (TKI) developed, was approved by the US Food and Drug Association (FDA) in 2003 to treat NSCLC, 22 followed by the FDA approval of erlotinib for NSCLC in 2004 23 and lapatinib for the treatment of ERBB2 positive breast cancer in 2007. 24However, resistance patterns to ERBB inhibitor treatment emerged due to secondary mutations at gatekeeper residues (e.g., T790M in EGFR) i.e., larger residues sterically impeded inhibitor binding. 25Furthermore, triggering alternative downstream signaling routes and the activation of bypass survival tracks via other RTKs also accounts for the acquired drug resistance to ERBB receptors. 26,27To avoid resistance, various third generation TKI drugs were developed, such as osimertinib targeting the sensitizing mutation EGFR-T790M to treat NSCLC. 28,29Furthermore, overactive ERBB4 is associated with reduced interneuron activity in the prefrontal cortex leading to an imbalance of excitation and inhibition, and consecutively to symptoms of schizophrenia. 30,31Thus, ERBB4 is regarded as a critical target for schizophrenia, and drug development is required to identify compounds that selectively target ERBB4, 32 but not EGFR, its most related receptor within the ERBB family.
As activated ERBB receptors can form homo or heterodimers restricted to their sub family, 2 targeting only one family member and modulating its downstream signaling is difficult.In addition to those challenges, the development of kinase inhibitors is challenging in general, as kinases are targeted by only 3% of all marketed drugs. 33Notably, kinases and especially RTKs, together with G protein-coupled receptors (GPCRs), represent one of the two most important drug targets in human cells. 33Biochemical binding assays and FRET-based assays are frequently used to profile compounds that interact with RTKs, including ERBB receptors, and offer valuable insights into the binding properties of various compounds.However, it is essential to recognize their limitations, particularly the lack of physiological context in test tube (in-vitro) settings, the inability to account for physiologically relevant interactions at the cell membrane (which is crucial for RTKs), and the potential artifacts from those in-vitro conditions. 34Standard reporter gene assays in living cells may solve some aspects of the limitations mentioned above.However, single assays are not able to deliver functionally diverse phenotypes from the very same cell population (i.e., an agonistic and antagonistic effect monitored on different pathways from one well).Therefore, the development of a holistic cell-based assay system that enables the monitoring of ERBB activities and downstream pathways may substantially contribute to the advancement of better therapeutic agents targeting ERBB receptors.
Here, we describe a barcoded profiling assay, termed 'ERBBprofiler', to simultaneously assess the activity of ERBB receptors in their native environment at the cell membrane and their downstream responses to key cellular pathways in living cells.Selective activities of individual ERBB receptors and pathway responses associated with receptor activation or inhibition can be monitored in the same well upon drug treatment.While ERRB receptor activities at the membrane were monitored by split TEV recruitment assays, pathway responses were measured by optimized nuclear pathway sensors.By profiling target selectivity and physiological responses of established drugs and compounds that are either approved, currently tested in clinical phases, or failed, we have demonstrated the capabilities of the cell-based assay platform to identify relevant features of ERBB antagonists.Lastly, we used the assay platform to profile novel ERBB4 selective antagonists, supporting the notion that the profiling assay can be used to accelerate early drug discovery campaigns.

Design of a barcoded ERBB family receptor and pathway profiling assay
Profiling of both ERBB targets and downstream pathway activities was performed by genetically encoded split TEV and pathway reporter gene assays, respectively, and requires a readout that is amenable to parallelization (Figure 1A).Therefore, the firefly luciferase reporter of each reporter plasmid was replaced by a unique DNA barcode that is transcribed into an RNA barcode, which can be isolated from cells and quantified by next-generation sequencing (NGS). 35,36For establishing the multiparametric ERBB receptor profiling assay, we first cloned small (n % 20) libraries of the reporter plasmids encoding (1) a barcoded Gal4-VP16 reporter for split TEV assays consisting of clustered upstream activating sequences (10xUAS), (2) a barcoded pathway sensor for MAP kinase signaling based on the early growth response 1 promotor (EGR1p sensor), (3) a barcoded pathway sensor for cAMP/calcium signaling based on cAMP response element (CRE sensor), (4) a barcoded pathway sensor for calcium signaling based on the response element of the nuclear factor of activated T-cells (NFAT sensor), and (5) a barcoded sensor for assessing baseline activities based on the major late promoter (MLP) from adenovirus.For the simultaneous monitoring of target and pathway activities, a subset of these barcode reporters (3 unique barcodes for UAS, 2 unique barcodes each for CRE, EGR1p, NFAT, and 1 for MLP) was transfected with plasmids encoding split TEV compatible ERBB receptors (i.e., EGFR, ERBB2 in combination with ERBB3, or ERBB4) and the split TEV RTK adapter 3xSH2-GRB2 37 into PC12 cells for ERBB receptor assays.In addition, we wished to add an unrelated target as a control assay.Thus, another subset of the reporter plasmids was transfected with the split TEV compatible serotonin receptor 2A (HTR2A), a Ga q -coupled GPCR, and the split TEV GPCR adapter ARBB2-CTEV 38 for an HTR2A assay (Figures S1A-S1F; Table S1).Therefore, each batch of cells contained the plasmids encoding a receptor, its cognate adapter, and the split TEV and pathway reporters with a set of unique barcodes enabling to assess the activities of one target, i.e., an ERBB receptor, or HTR2A, and its downstream signals.Multiplexing of target and pathway assays was then further conducted by mixing cell batches into one well and continued at the plate level.Thus, barcoding occurred at the levels of single assays, wells, and plates, and enabled the generation of thousands of data points from one ERBBprofiler experiment (Figure 1B).In a typical profiling experiment, batches of transfected PC12 cells were mixed, plated onto 24-well plates, cultured for 20 h, starved for 16 h in medium with reduced serum conditions, and treated with increasing doses of epidermal growth factor (EGF, ligand for EGFR), EGF-like domain (EGFld, ligand for ERBB3 and ERBB4), and serotonin (ligand for HTR2A) for 6 h before lysis (Figure S1G).To proceed to the sequencing of barcodes using next-generation sequencing and to reduce handling, a unique DNA barcode oligo, termed well barcode, was added to each lysate originating from one well, enabling the tracking of individual lysates in a pool.Next, all lysates from one 24-well plate were pooled for combined processing, a process called 'Tag&Pool' (read 'Tag-and-Pool', see STAR Methods) (Figures S1H  and S1I).

Selectivity of ERBB receptor activation, HTR2A activation, and their downstream signaling by single ligands correlate in barcoded assays
In the initial ERBBprofiler assay experiment, we examined the impact of EGF, EGFld, and serotonin on the activation on ERBB receptors, HTR2A, and their key downstream pathways.While EGF treatment selectively stimulated the activity of EGFR, addition of EGFld led to the activation of ERBB2/3 and ERBB4 (Figure 2A).The responsiveness to the ligands was clearly observed in dose-response graphs for EGFR (Figure 2B) and ERBB4 (Figure 2C).PC12 endogenously express EGFR, and EGFR stimulation leads to the activation of the ERK1/2 branch of MAPK signaling. 39In our cellular profiling assay, this branch of MAPK signaling was captured by the EGR1p sensor.As expected, PC12 cells that were transfected with the EGR1p sensor only responded to increasing EGF concentrations (Figures 2A and 2D).Conversely, EGFld treatment did not lead to any response.When ERBB4 was co-transfected with the EGR1p sensor, EGFld indeed induced an EGR1p response, as A B Figure 1.Design of a fully integrated, multiplexed assay to profile activities of ERBB receptor family and physiological signaling in parallel (A) Summary schematic showing target and pathway assays of the barcoded ERBBprofiler assay.Activities of receptors were monitored using split TEV assays, while activities of pathways were measured using pathway sensors.BC, barcode.Upper inset: targets used in this study.Lower inset: Barcode reporters for targets (UAS), pathways (EGR1p for MAPK/ERK signaling, CRE for cAMP/Ca 2+ signaling, and NFAT for Ca 2+ signaling), and a control (MLP).(B) Workflow for molecular barcoding of assays, wells, and plates.On the first level of barcoding, 50 assays, each with a unique barcode, are monitored in one well.On the second level, the lysates of the wells are barcoded, and on the third level, the plates are barcoded.Barcodes are sequenced using next generation sequencing (NGS), and data are analyzed.In a typical experiment comprising ten 24-well plates, 12,000 data points are generated from ten Tag&Pool (a method to process 24 lysates combined, see STAR Methods section) samples.See also Figure S1 and Table S1.
evident from an increase in EGR1p sensor activity (Figures 2A and 2E).Serotonin treatment selectively activated HTR2A, and physiologically linked calcium signaling was induced when HTR2A was activated, as indicated by increased CRE and NFAT reporter activities (Figures 2A and  2F-2H).EGF treatment led to a low activation of the NFAT sensor in the presence of co-transfected HTR2A, a finding that can be explained by crosstalk between EGFR and HTR2A. 40Notably, neither EGF nor EGFld activated CRE and NFAT sensors in the absence of HTR2A, nor did serotonin lead to the activation of the EGR1p sensor, supporting the notion that the stimuli were selective for both targets and physiological pathways.To benchmark the performance of barcoded assays, target and pathway assays for EGFR, ERBB4, and HTR2A were replicated with a luciferase-based readout at dose response (Figures 2B-2H).Barcoded and luciferase assays showed comparable dose-response curves and EC 50 values for each assay tested.Responses to target activation (measured by split TEV) and cellular signaling (measured by pathway sensors) correlated for both barcoded assays and luciferase assays (see Figure S2).In addition, cells were treated with a mix of all three ligands to assess any potential synergistic effects among ligands, as this mix of ligands was planned to be applied in antagonist profiling assays to reduce sample numbers.Notably, the effects observed by the individual stimuli and the mix matched for both targets and pathway assays (Figures 2A and S2), enabling a simultaneous assessment in compound profiling assays.All EC 50 values for barcoded assays are summarized in Table S2.(B-H) Barcoded assays align with luciferase readouts.Visualization of selected data from (A), comparing barcoded assays (black) with luciferase assay readouts (red).Assays for receptors were performed using split TEV, assays for signaling pathways with pathway sensors.Dose response graphs for EGFR (B), ERBB4 (B), EGR1p only (D), and EGR1p and ERBB4 transfected (E), HTR2A (F), CRE and HTR2A transfected (G), and NFAT and HTR2A transfected (H) with single stimuli applied at increasing concentrations.EGFld, EGF-like domain.Error bars represent SEM, n = 3 for barcoded assays, and n = 6 for luciferase assays.See also Figure S2 and Table S2.5][46] Clozapine was selected as an approved drug for targeting HTR2A. 47As antagonist assays require activated receptors and pathways, we selected the mix of ligands to stimulate ERBB receptors and HTR2A, as well as signaling pathways.As receptors and pathway sensors were sufficiently activated at a concentration of 30 ng/mL EGF, 10 ng/mL EGFld, and 1 mM serotonin (Figure 2A), dose response treatments for all selected drugs were performed in PC12 cells using those agonist concentrations and the same experimental setup as outlined before.Agonists were applied as a mix to reduce sample number.To further simplify handling, antagonist and agonist treatments were conducted simultaneously (Figure S1G).From a total of 240 samples, batches of 24 samples were pooled at the lysis stage using the Tag-and-Pool process to produce ten samples that were processed for barcode sequencing, yielding 12000 data points in total (Figure 1B).For each compound, we obtained a dose-response dataset for each receptor (EGFR, ERBB2/3, ERBB4, and HTR2A) measured by split TEV assays and pathway sensors (EGR1p for the ERK branch of MAPK signaling, CRE for cAMP/calcium signaling, NFAT for calcium signaling) linked to each receptor (Figures 1A and 3A).
When comparing our results to IC 50 values of phospho-blots and cell proliferation studies, we either had comparable potency values (e.g., for AG1478 on EGFR, 51 erlotinib on phospho-ERK, 52 osimertinib on ERBB4, 28 poziotinib on EGFR and ERBB2/3, 53 and TAS6417 on EGFR 48 ) or an order of magnitude higher IC 50 values, possibly due to the use of different cell lines and assay readouts (e.g., for erlotinib on EGFR, gefitinib on EGFR and ERBB4, 54,55 osimertinib on EGFR, 56 and pyrotinib on EGFR. 49][59] Of note, many ERBB antagonists, including osimertinib, poziotinib, and pyrotinib, caused an upregulation of the CRE sensor at high compound concentration of 10 mM.An increased CRE sensor activity may pinpoint to both increased cAMP and calcium concentrations that may arise from cellular stress. 60To validate the results from the barcoded profiling assay, we conducted single luciferase assays using the CRE sensor as readout for these three compounds and AG1478, as the latter did not cause any increased CRE sensor activities in the barcoded assay.In PC12 cells that were transfected with the CRE sensor only, we found similar patterns of CRE sensor activation at high compound concentrations of osimertinib, poziotinib, and pyrotinib, but not for AG1478 (Figures 3F-3I), suggesting a cell intrinsic response caused by a subset of inhibitors.To identify whether the CRE sensor responded to increased cAMP or calcium concentrations, we conducted orthogonal assays in PC12 cells to measure cAMP using a GloSensor assay, while calcium was assessed using Fluo-4 a.m., a cell permeable calcium indicator.AG1478 treatment did not cause any increase in neither cAMP nor calcium (Figure 3J), while higher concentrations (R3 mM) of osimertinib (Figure 3K), poziotinib (Figure 3L), and pyrotinib (Figure 3M) resulted in elevated cAMP, but not calcium levels.(F-I) Dose response graphs for CRE sensor responses in PC12 cells using luciferase as readout for AG1478 (F), osimertinib (G), poziotinib (H), and pyrotinib (I).In addition to the increasing concentrations of the compounds shown, all assays contained the constant stimulation mix as in (A).(J-M) Dose response graphs for calcium and cAMP assays using Fluo-4 a.m. and GloSensor, respectively, as readouts in PC12 cells treated with increasing concentrations of AG1478 (J), osimertinib (K), poziotinib (L), and pyrotinib (M).As in luciferase assays, the constant stimulation mix was constantly present, next to the mentioned compounds.Error bars represent SEM, n = 3 for barcode assays (B-E), n = 6 for luciferase, Fluo-4 a.m., and GloSensor assays (F-M).See also Figure S3, Tables S3 and S4.

Pyrotinib reveals selectivity for ERBB4 over EGFR
A key feature of the established ERBB profiling assay should be that it can discriminate between EGFR and ERBB4 selective compounds.Next, we aimed to validate the barcode assay results for AG1478 and pyrotinib using single luciferase assays and Western blotting as orthogonal assays.AG1478 inhibited EGFR over ERBB4 in split TEV luciferase assays (Figure 4A).However, this preference for EGFR was not present anymore when the EGR1p pathway sensor was used as readout (Figure 4B), indicating that a direct coupling of the reporter to the target, as in split TEV assays, is required to detect subtle differences.To confirm AG1478's activity on EGFR over ERBB4, A549 cells, which endogenously express EGFR, 61 and T-47D cells, which endogenously express ERBB4, 37,62 were treated with constant concentrations of agonists (i.e., EGF for A549 cells, and EGFld for T-47D cells) and increasing concentrations of AG1478.Indeed, AG1478 preferentially antagonized phospho-EGFR over phospho-ERBB4 in Western blot analyses (Figures 4C-4F).In addition, AG1478 inhibited EGFR mediated downstream ERK signaling, a finding that was quantified in cellulo for the first time by our barcoded assay (Figures 3A-3C).These results were validated by the luciferase assay using the EGR1p sensor as well as biochemically by assessing phospho-ERK1/2 in a Western blot analysis (Figures 4B and 4F).In sum, the preference for AG1478-mediated EGFR over ERBB4 inhibition was present at receptor level but was lost at pathway level in both the barcoded assays and Western blot assays.
Conversely, pyrotinib preferentially inhibited ERBB4 activity over EGFR activity in split TEV receptor assays (Figure 4G).However, pyrotinib's preference for ERBB4 was compromised in pathway assays using the EGR1p sensor as downstream readout (Figure 4H).To confirm pyrotinib's activity on ERBB4 over EGFR, we treated A549 cells and T-47D cells with constant concentrations of agonists (i.e., EGF for A549 cells, and EGFld for T-47D cells) and increasing concentrations of pyrotinib.Indeed, pyrotinib treatment led to a more potent inhibition of ERBB4   (A and B) Dose response assays comparing AG1478 selectivity for receptors EGFR and ERBB4 (A) and downstream MAPK signaling (B) using firefly luciferase assays in PC12 cells.Assays for receptors were performed using split TEV, assays for MAPK signaling were conducted with an EGR1p pathway sensor.In addition to the increasing concentrations of AG1478, EGFR and ERBB4 assays contained a constant concentration of EGF (30 ng/mL) or EGF-like domain (10 ng/mL), respectively.(C and D) Western blot analyses of p-EGFR (in A549 cells) (C) and p-ERBB4 (in T-47 cells) (D) using increasing concentrations of AG1478.(E and F) Quantification of (C) and (D).(G and H) Dose response assays comparing pyrotinib selectivity for receptors EGFR and ERBB4 (G) and downstream MAPK signaling (H) using firefly luciferase assays in PC12 cells.Assays were conducted as in (A, B). (I and J) Western blot analyses of p-EGFR (in A549 cells) (I) and p-ERBB4 (in T-47 cells) (J) using increasing concentrations of pyrotinib.(K and L) Quantification of (I) and (J).Error bars represent SEM, n = 6 for luciferase assays (A, B, G, H), n = 3 for Western blot assays (E, F, K, L). activity as measured by phospho-ERBB4 and phospho-EGFR (Figures 4I-4L).Similar to the barcoded and luciferase EGR1p pathway assays, EGFR and ERBB4-dependent phospho-ERK1/2 signals did not substantially differ in pyrotinib treated cells (Figures 4H and 4L).Taken together, the barcoded ERBB profiling assay can assess antagonistic actions on ERBB receptors and key physiological pathways, assess a compound's selectivity, and identify potential side effects in one experiment.

Discovery of novel ERBB4 selective antagonists
Previously, we identified spironolactone as ERBB4 antagonist in a drug repurposing screen, while spironolactone's metabolite canrenone did not inhibit ERBB4 activity. 32To test the capabilities of the barcoded ERBBprofiler assay, we treated PC12 assay cells with increasing concentrations of both spironolactone and canrenone and agonists.Results confirmed the antagonistic effect of spironolactone on ERBB4 with similar activity (IC 50 : 4.327 mM) (Figure S4).Conversely, canrenone did not inhibit any ERBB receptor nor pathway monitored.Furthermore, we found that spironolactone inhibited HTR2A at 10 mM, but not at lower concentrations (Figure S4).
Next, we sought to profile potentially selective ERBB4 antagonists, termed compounds A and B, that were designed and synthesized in a proprietary kinase inhibitor program to challenge our barcoded ERBB family profiling assay.The main goal was to identify compounds that show a selectivity for ERBB4 over EGFR for the potential use in follow-up drug discovery campaigns.Both compounds A and B more efficiently inhibited ERBB4 than EGFR (Figure 5A).When extracting the data from this heatmap and computing dose-response curves, we found that both compounds showed a higher activity for ERBB4 than for EGFR and to some extent for ERBB2/3 (Table S3).Importantly, compound B had both better efficacy and preference for ERBB4 over EGFR (ERBB4 IC 50 : 0.142 mM, EGFR IC 50 : 1.085 mM, about 8-fold) than compound A (ERBB4 IC 50 : 0.575 mM, EGFR IC 50 : 1.146 mM, about 2-fold) (Figures 5B and 5C), defining compound B as frontrunner compound.Similarly, ERK signaling was inhibited by these two compounds in an EGFR, ERBB2/3, and ERBB4 mediated manner, reflecting the inhibition at receptor level (Figures 5D and 5E).Biochemical validation by Western blotting of phospho-EGFR (in A549 cells) (Figure 5F) and phospho-ERBB4 (in T-47D cells) (Figure 5G) confirmed that compound B preferentially antagonized ERBB4, as revealed by quantification of phosphorylation levels (Figure 5H).An analysis of downstream phospho-ERK1/2 showed that compound B reduced the ERK pathway activity to about 70% of activated EGFR and ERBB4 (Figure 5I).To confirm that compounds A and B inhibited EGFR and ERBB4 at different potencies, we conducted LANCE kinase activity assays.In this in vitro executed enzymatic assay, synthetic substrates are incubated with EGFR and ERBB4 purified protein, and the amount of phosphorylated substrate is detected by a specific anti-phosphopeptide antibody, resulting in a time resolved fluorescence resonance energy transfer (TR-FRET) signal.Notably, both compounds A and B inhibited ERBB4 more effectively than EGFR (Figures 5J and 5K).In addition, compound B displayed a better preference profile for ERBB4 than EGFR, as evident from IC 50 values for compound B (ERBB4: 0.059, EGFR: 0.525) and compound A (ERBB4: 0.01, EGFR: 0.067).Thus, compound B showed an about 9-fold preference for ERBB4, while compound A only displayed an about 7-fold preference, validating the findings of our cellular profiling assay that compound B had better selectivity for ERBB4.
An analysis of downstream pathway activities further showed that compound A treatment at 10 mM led to the activation of the CRE sensor for all targets tested (Figure 5A).A separate validation assay in PC12 cells by transfecting only a CRE sensor linked to luciferase readout confirmed this opposite behavior of these two novel ERBB4 antagonists.In this assay, PC12 cells were treated with increasing concentrations of either compound A or B and the stimulation mix containing EGF, EGFld, and serotonin to mimic the conditions from the barcoded assays.While compound A treatment led to increased CRE activity at concentrations of R3 mM, compound B treatment inhibited CRE activity in a dose-dependent manner (Figure 5L).To assess whether the CRE sensor is modulated by altered levels of cAMP or calcium in response to ERBB4 antagonist treatment, the GloSensor assay was used to measure cAMP and Fluo-4 a.m. to assess calcium.As in luciferase validation assays, PC12 cells were treated with increasing concentrations of either compound A or B and the stimulation mix.Calcium levels were neither affected by compound A nor by compound B. However, compound A concentrations of R3 mM caused an increase in cAMP levels, while compound B treatment, starting with 1 mM and higher, caused a steady decrease in cAMP, mirroring the effects from the CRE sensor barcode and luciferase assays (Figures 5M and 5N).Furthermore, treatment with compound B, but not compound A, resulted in an inhibition of HTR2A at target (split TEV assay) and pathway levels (CRE assay) in PC12 cells (Figures 5A, S5A, and S5B), a finding that was validated by independent luciferase assays for compound B (Figure S5C).Taken together, we identified two novel ERBB4 selective antagonists, with compound B being more selective and, at least in cell culture, more potent than compound A.

DISCUSSION
We describe the establishment of a barcoded ERBB receptor profiling assay that enables the simultaneous profiling of compound actions on ERBB targets and key downstream pathways in living cells.Specifically, split TEV assays for monitoring the activities of full-length EGFR, ERBB2/3, ERBB4, and HTR2A (a GPCR as non-ERBB receptor control) are parallelized with pathway assays for cAMP/Ca 2+ signaling (CRE sensor), pure Ca 2+ signaling (NFAT sensor), and the ERK branch of MAPK signaling (EGR1p sensor) in this multiparametric assay platform.Notably, downstream pathway responses were uniquely assigned to each receptor, allowing the identification of functionally distinct activities when treated with a compound from one well.For example, while the MAPK sensor EGR1p was inhibited by pyrotinib, the CRE sensor was activated at high concentrations for all ERBB receptors.Furthermore, varying intensities of inhibition or activation for one pathway were clearly assigned to a specific ERBB receptor activity.Barcoded assays are as responsive and robust as single luciferase assays, as evidenced by similar EC 50 values obtained from these two assay types for targets and pathways tested.Through applying a multilevel barcoding strategy for analyzing cellular activities (i.e., sensors with unique barcodes for targets and pathways), wells (sample barcoding through pooling of cell lysates using Tag&Pool and PCR-based barcoding), and cell culture plates (PCR-based barcoding) in parallel, we reduced sample numbers and hands-on time for sample processing and NGS analyses, while processing thousands of data points.In addition, we reduced sample numbers in drug profiling assays even further by stimulating ERBB receptor and HTR2A targets with a ligand mix.The principle of the barcoded profiling assay can be extended in the future to any other RTK, GPCR, or other targets for which split TEV assays are available or can be developed.Pathway sensors used here can be applied to any RTK or GPCR assay, and additional sensors can be developed to capture activities of other cellular pathways.However, the number of pathway sensors possibly used per target is limited by the well size (i.e., number of cells that fit into a well), the number of targets used per well, and the depth of the sequencing, thus affecting the overall assay's complexity.
Commonly used kinase profiling assays for ERBB receptors are based on biochemical kinase platforms, which can be divided into two classes, activity assays and binding assays.Activity assays directly or indirectly measure the catalytic product and include radiometric platforms (e.g., HotSpot), 63 luminescence-based (e.g., Kinase-Glo), and fluorescence-based platforms (e.g., Lance TR-FRET assay or LanthaScreen TR-FRET assay). 64(Note that compounds A and B were validated using the Lance TR-FRET kinase activity assay.)In contrast, binding assays measure the binding of compounds to the kinase active site, but not the catalytic product (e.g., KinomeScan). 64All of these are single assays and , and downstream signaling pathways in PC12 cells.Assays for receptors were performed using barcoded split TEV assays, assays for signaling pathways using barcoded pathway sensors.In addition to the increasing concentrations of biochemical in nature.A more focused platform for the analysis of RTK activities uses a membrane-based sandwich immunoassay with phosphotyrosine antibodies and some degree of multiplexing as one cellular lysate can be used to profile 49 different RTKs. 65RTKs and cellular downstream effectors can also be profiled using an image-based approach, as described for EGFR. 66This method was reported to be robust but rather labor intensive, making multiplexing inefficient.Although most of these assays use smaller well formats than we did in this study, multiplexing at the assay level (including both targets and pathways), well level, and plate level as introduced for our multilevel barcoded reporter profiling assay is not feasible.In contrast, using NGS as a readout for our barcoded assay, we simultaneously monitored the effects of compounds on ERBB receptors and their downstream pathways, using 50 assays per well and obtaining 12,000 data points from a standard assay setup.We challenged the ERBB receptor profiling assay with eight different ERBB receptor antagonists (AG1478, erlotinib, gefitinib, lapatinib, osimertinib, poziotinib, pyrotinib, and TAS6417) that were developed to target different types of cancers.All these drugs inhibited the activity of EGFR, ERBB2/3, ERBB4 at different levels, and likewise, canonical downstream ERK signaling.When systematically comparing our IC 50 data with published data obtained from cellular assays, we noticed that we had comparable potencies for some of the eight ERBB antagonists on selected targets and pathways, 28,48,[51][52][53] while for other combinations our potency values were an order of magnitude higher (cf.5][56] The latter case may be due to different cell types and/or assay systems applied.Nevertheless, a substantial number of studies used phospho-blots to demonstrate the drug effect on ERBB receptors.Although this is a very reliable technique, due to its technical demands, most of these findings were only qualitative in nature, especially when studying pathway activities.Conversely, our ERBB profiling assay allows the monitoring of multiple compounds on ERBB receptors and linked pathways in parallel, saving both hands-on time and money.We validated our previous findings for lapatinib that showed a preference for ERBB2/3 and ERBB4 over EGFR, 37 while spironolactone was an ERBB4 selective antagonist. 32Most strikingly, AG1478 displayed a preference for EGFR, while pyrotinib showed a preference for ERBB4.As this was expected for AG1478, 67 pyrotinib's effect on ERBB4 is novel.Together, these features verify that the barcoded ERBB receptor profiling assay can detect both preferred activities of compounds for EGFR versus ERBB4 and represents an optimal route to assess ERBB antagonists in living cells.The ERBBprofiler assay enables the simultaneous monitoring of target and pathway effects in living cells.In this respect, we identified pathway characteristics for AG1478, as we quantified its effect on ERBB4 and MAPK signaling downstream of EGFR for the first time in cellulo.Likewise, we monitored effects of TAS6417 on ERBB2/3 and ERBB4 for the first time in cellulo. 57,68,69Poziotinib was the only covalent inhibitor we tested, and it is known to inhibit ERBB receptor activities at nanomolar range. 53,70Indeed, we observed a very strong activity of this drug, both at target and pathway level, arguing that our platform reliably detects compound effects across several magnitudes of molar range. Frequently, we observed enhanced potency of drugs on pathway assays (i.e., lower IC 50 values) when compared to target assays, suggesting that compound effects are amplified within the process of cellular signaling. 71Conversely, target assays were more useful in identifying a drug's selectivity for a given ERBB receptor.Importantly, activity trends for targets aligned well with pathway activities in our profiling assay, supporting the notion that both assay types can be used to assess compound actions.Nevertheless, for some conditions, drug effects on targets may be very subtle or even hidden, 72,73 and pathway assays may be used to unveil these effects through cellular signal amplification.
At high drug concentrations of 10 mM, we observed an upregulated CRE sensor response for many of the tested drugs (i.e., erlotinib, gefitinib, lapatinib, osimertinib, poziotinib, pyrotinib, TAS6417, canrenone, and compound A).Using orthogonal assays specific for cAMP and calcium we found that high concentrations of poziotinib treatment led to an increase of cAMP.Increased cAMP concentrations are linked to cellular stress pathways, cellular energy status, as well as oxidative and proteotoxic stress, 14,[74][75][76] suggesting that high concentrations of selected ERBB antagonist are the cause for this abnormal cellular signature.Remarkably, clozapine, spironolactone, and compound B did not activate the CRE sensor at 10 mM, suggesting that these compounds are also better tolerated at higher, albeit non-physiological, concentrations.In fact, PC12 cells treated with compound B, and without transfecting any other plasmids than the GloSensor, resulted in a concentration-dependent decrease of cAMP, which might be mediated by antagonizing activities conferred by HTR6A, a Ga s protein coupled GPCR expressed in PC12 cells, 77 or by inhibiting components of the protein kinase A/cAMP pathway.
Increased ERBB4 activity leads to an excitation/inhibition imbalance in schizophrenic patients, 30,31 and antagonizing ERBB4 activity using spironolactone improved schizophrenia relevant phenotypes including cognition in a Nrg1 transgenic mouse model, 32 prompting also a clinical trial with spironolactone as adjuvant to antipsychotic medication, i.e., co-administering spironolactone with an established antipsychotic, e.g., risperidone or aripiprazole. 78Notably, a separately conducted clinical trial using spironolactone as add-on therapeutic to risperidone reported improvements for positive and negative symptoms, but not cognitive deficits. 79In addition, spironolactone as mineralocorticoid receptor antagonist was shown to improve cognition in mouse models of type II diabetes 80 and Alzheimer's disease. 81In our profiling assay, we found that spironolactone also inhibits HTR2A, and antagonists to HTR2A are frequently used for the treatment of schizophrenia, 82 suggesting that such a polypharmacological profile may be beneficial for alleviating symptoms in schizophrenic patients.
When targeting ERBB4, at least in the CNS, a drug's selectivity over EGFR is most crucial, as EGFR activity is key for the propagation of neural precursors and the differentiation of these into neurons, while ERBB4 is linked to schizophrenic phenotypes. 83,84In addition, ERBB4 is most closely related to EGFR. 85,86A recent study reported two investigational compounds that are ERBB4-selective over EGFR, with a 2.5-fold pref.87.However, compounds with a higher selectivity are required to bypass any non-desired effects that may be mediated through EGFR inhibition.Here, we identify two novel compounds that were synthesized in a proprietary kinase inhibitor program and that have an increased selectivity for ERBB4 over EGFR, with compound B displaying an about 8-fold preference.Importantly, this strong preference for ERBB4 for compound B we observed in the cellular profiling assay was confirmed by in-vitro kinase assays, albeit to a lesser degree.Furthermore, compound B, but not compound A also inhibited HTR2A.Therefore, compound B with its pharmacological profile may represent a lead structure

Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Michael Wehr (michael.wehr@med.uni-muenchen.de).Luciferase reporter assays 50,000 PC12 cells were seeded per 96-well onto poly-L-lysin (PLL)-coated plates and transfected the following day (day in vitro 1, DIV1) with assay plasmids using Lipofectamine 3000 (Thermo Fisher Scientific).For split TEV assays, plasmids (pcDNA3 or pTag4C backbone) encoding receptor-NTEV-tcs-GV, GRB2-SH2-CTEV (for ERBB receptors) or ARBB2-1-383-CTEV (for HTR2A) (all 10 ng/well), and the p10xUAS-luc2 reporter plasmid (10 ng/well) were used.For pathway assays, the receptor plasmids (15 ng/well) and pathway reporter plasmids (15 ng/well) pEGR1p-luc2 for monitoring MAPK signaling, pCRE-CMVmin-luc2 for monitoring cAMP/Ca 2+ signaling, or p6xNFAT-CMVmin-luc2 for Ca 2+ signaling were used.Combinations of assays conducted are summarized in Table S5.Transient transfections were conducted according to the manufacturer's instructions.On DIV2, cells were starved in medium containing 1% FCS for 16 h.On DIV3, a stimulus was added at increasing doses or, for antagonist assays, at single concentrations (30 ng/mL EGF for activating EGFR; 10 ng/mL EGFld for activating ERBB2/3 and ERBB4; and 1 mM serotonin for activating HTR2A) for 6 h.In the case of antagonist assays, compounds were added at increasing doses together with the stimulus.Cells were lysed in 1x passive lysis buffer (Promega) and subjected to a firefly luciferase assay using a selfmade substrate for firefly luciferase. 100Luciferase activity was analyzed in a Mithras LB 940 Multimode Microplate Reader (Berthold Technologies).All assays were run in 96-well plates using six replicates per condition.

GloSensor cAMP assay
The GloSensor cAMP assay was conducted with the same parameters (50,000 PC12 cells per 96-well) as described above for the firefly luciferase assay, with however, three differences.Specifically, (1) 30 ng/well of the pGloSensor-22F plasmid (Promega, E2301) was used for transfection, and (2) on DIV3, cells were equilibrated in assay medium containing a 2% v/v dilution of D-luciferin (Synchem, bc219) for 2 h at 37 C before (3) the treatment of compounds for 15 min and lysis in 1x passive lysis buffer (Promega) and luciferase activity measurement.
Fluo-4 a.m.calcium assays 50,000 PC12 cells were seeded per 96-well onto poly-L-lysin (PLL)-coated clear bottom plates (PerkinElmer, 6055302) and starved on DIV1 for 24 h.On DIV2, cells were pre-equilibrated in 10 mM Fluo-4 a.m.(Thermo Fisher Scientific, F14201) away from light for 1 h at 37 C. Next, cells were incubated in the indicator-free starvation medium to allow de-esterification for 20 min away from light at 37 C. Background fluorescence was then measured using the BMG POLARstar Optima Microplate Reader prior to adding the treatments and continuously measuring the fluorescence for the next 30 min.Fluorescence was measured using the bottom optics.

Barcode reporter assays Transfection of reporter plasmids and stimulation conditions
Mulitplexed barcoded reporter assays were conducted in PC12 cells using 24-well plates, with 3 replicates per condition.Per well, 250,000 PC12 cells were transfected in solution with assay plasmids using Lipofectamine 3000.Per well, 5 batches of transfected cells were used.Per well, amounts of assay plasmids were 33 ng of target and adapter plasmids, and 27 ng of each reporter plasmid (note that 10 reporters were used per batch), totaling to 336 ng/well.Combinations of barcoded assays conducted are summarized in Table S6, barcode sequences used as RNA reporters are listed in Table S1.Transfected cells were incubated for 2 h at 37 C and 5% CO 2 .Tubes (15 mL or 50 mL Falcon tubes) containing transfected cells were loosely closed to allow the CO 2 flow and put tilted at 45 angle to enhance transfection efficiency and viability.Cells were then centrifuged for 5 min at 1000 rpm, the whole supernatant was carefully aspirated, and cells were plated in maintenance medium into PLL-coated 24-well plates for 24 h.Cells were then starved for 16 h as described above and treated for 6 h with compounds.Agonists and antagonists were applied simultaneously.Cells were lysed in 400 mL/well in a wash and lysis buffer (100 mM Tris/HCl pH 7.5, 500 mM LiCl, 10 mM EDTA, 5 mM DTT, 1% LiDS).Plates were shaken for 10 min at 200 rpm for thorough lysis.

Isolation of barcodes, library preparation, and next-generation-sequencing
For Tag&Pool, a procedure to combine multiple cell lysates for single purification and processing of barcode reporter RNAs, second level barcodes (to track wells, see Figures 1 and S1I) were added to the lysates to a final concentration of 0.125 mM for annealing at 65 C for 15 min.Once cooled to room temperature, all 24 lysates from one 24-well plate and 20 mL of M-PVA OdT2 beads (Chemagen, Cat.No. CMG-231) were pooled.Next, beads were subjected to a series of wash steps, including one wash with 100 mL of 1x High-Capacity reaction buffer (High-Capacity cDNA Reverse Transcription Kit, Thermo Fisher Scientific, Cat.No. 4368814).Finally, cDNA synthesis from beads was performed in 20 mL of cDNA High-Capacity cDNA Reverse Transcription mix at 25 C for 25 min.For barcoded ('tagged') cDNA amplification, a forward primer containing the Read1 Illumina adapter sequence and an UMI sequence was used, in combination with a Read2 reverse primer (30 PCR cycles).Illumina indices and sequencing adapters were attached (10 PCR cycles), the final barcode libraries were pooled in equimolar ratio (2 p.m.) and subjected to paired end, dual index sequencing with the NovaSeq 6000 SP Reagent Kit v1.5 (Illumina GmbH, Cat.No. 20028401 or 20040719).Oligos used are listed in Table S7.

Biochemistry
Phosphorylation levels of EGFR were assessed 37 in A549 cells, while phosphorylation levels of ERBB4 were assessed in T-47D cells.Before stimulation experiments were performed, both cell types were starved overnight in 1% FCS and compounds were incubated for 1 h.A549 were stimulated with 30 ng/mL EGF for 5 min, while T-47D cells were stimulated with 10 ng/mL EGFld for 5 min.For lysis, cells were washed

Figure 2 .
Figure 2. Selectivity of receptor activation and downstream signaling by single ligands correlate in barcoded assays (A) Heatmap showing stimulation profiles on ERBB receptors, HTR2A, and downstream signaling pathways.Assays for receptors were performed using barcoded split TEV, assays for signaling pathways with pathway sensors coupled to barcodes.Compound effects are shown as log2-transformed fold change.(B-H)Barcoded assays align with luciferase readouts.Visualization of selected data from (A), comparing barcoded assays (black) with luciferase assay readouts (red).Assays for receptors were performed using split TEV, assays for signaling pathways with pathway sensors.Dose response graphs for EGFR (B), ERBB4 (B), EGR1p only (D), and EGR1p and ERBB4 transfected (E), HTR2A (F), CRE and HTR2A transfected (G), and NFAT and HTR2A transfected (H) with single stimuli applied at increasing concentrations.EGFld, EGF-like domain.Error bars represent SEM, n = 3 for barcoded assays, and n = 6 for luciferase assays.See also FigureS2and TableS2.

Figure 3 .
Figure 3.The barcoded ERBBprofiler reveals known and previously uncharacterized selectivity properties of ERBB receptor antagonists (A) Heatmap showing antagonistic effects of compounds on ERBB receptors, HTR2A, and downstream signaling pathways in PC12 cells.Assays for receptors were performed using barcoded split TEV assays, assays for signaling pathways with barcoded pathway sensors.In addition to the increasing concentrations of the compounds shown, all assays contained constant concentrations of EGF (30 ng/mL), EGF-like domain (10 ng/mL), and serotonin (1 mM).Compound effects are shown as log2-transformed fold change.(B-E) Dose response graphs comparing drug selectivity for receptors EGFR and ERBB4 (B, D) and downstream MAPK signaling (C, E) of compounds AG1478 (B, C), and pyrotinib (D, E).Data was extracted from the heatmap shown in (A).n = 3.

Figure 3 .
Figure 3. Continued (F-I) Dose response graphs for CRE sensor responses in PC12 cells using luciferase as readout for AG1478 (F), osimertinib (G), poziotinib (H), and pyrotinib (I).In addition to the increasing concentrations of the compounds shown, all assays contained the constant stimulation mix as in (A).(J-M) Dose response graphs for calcium and cAMP assays using Fluo-4 a.m. and GloSensor, respectively, as readouts in PC12 cells treated with increasing concentrations of AG1478 (J), osimertinib (K), poziotinib (L), and pyrotinib (M).As in luciferase assays, the constant stimulation mix was constantly present, next to the mentioned compounds.Error bars represent SEM, n = 3 for barcode assays (B-E), n = 6 for luciferase, Fluo-4 a.m., and GloSensor assays (F-M).See also FigureS3, TablesS3 and S4.

Figure 4 .
Figure 4. Pyrotinib reveals selectivity for ERBB4 over EGFR (A and B) Dose response assays comparing AG1478 selectivity for receptors EGFR and ERBB4 (A) and downstream MAPK signaling (B) using firefly luciferase assays in PC12 cells.Assays for receptors were performed using split TEV, assays for MAPK signaling were conducted with an EGR1p pathway sensor.In addition to the increasing concentrations of AG1478, EGFR and ERBB4 assays contained a constant concentration of EGF (30 ng/mL) or EGF-like domain (10 ng/mL), respectively.(C and D) Western blot analyses of p-EGFR (in A549 cells) (C) and p-ERBB4 (in T-47 cells) (D) using increasing concentrations of AG1478.(E and F) Quantification of (C) and (D).(G and H) Dose response assays comparing pyrotinib selectivity for receptors EGFR and ERBB4 (G) and downstream MAPK signaling (H) using firefly luciferase assays in PC12 cells.Assays were conducted as in (A, B). (I and J) Western blot analyses of p-EGFR (in A549 cells) (I) and p-ERBB4 (in T-47 cells) (J) using increasing concentrations of pyrotinib.(K and L) Quantification of (I) and (J).Error bars represent SEM, n = 6 for luciferase assays (A, B, G, H), n = 3 for Western blot assays (E, F, K, L).

3 IFigure 5 .
Figure 5.The barcoded ERBBprofiler reveals novel ERBB4 selective antagonists (A) Heatmap showing antagonistic effects of LDC compounds on ERBB receptors, HTR2A, and downstream signaling pathways in PC12 cells.Assays for receptors were performed using barcoded split TEV assays, assays for signaling pathways using barcoded pathway sensors.In addition to the increasing concentrations of the compounds shown, all assays contained constant concentrations of EGF (30 ng/mL), EGF-like domain (10 ng/mL), and serotonin (1 mM).(B-E) Dose response graphs comparing drug selectivity for ERBB4 over EGFR (measured with split TEV) (B, C) and downstream MAPK signaling (measured with the EGR1p sensor) (D, E) of compound A (B, D), and compound B (C, E).Data was extracted from the heatmap shown in (A).(F-I) Orthogonal validation for compound B using Western blot analyses of p-EGFR and p-ERK1/2 (measured in A549 cells) (F) and p-ERBB4 and p-ERK1/2 (measured in T-47 cells) (G) using increasing concentrations of compound B (Cpd B). (H) Quantification of relative p-EGFR and p-ERBB4 from (F, G). (I) Quantification of relative p-ERK1/2 from (F, G). (J and K) In vitro kinase activity assays using LANCE assays for compound A (J) and compound B (K) showing dose response graphs comparing drug selectivity for ERBB4 (red) and EGFR (black).(L) Dose response graphs for CRE sensor responses in PC12 cells using luciferase as readout for compounds A (black) and B (red).In addition to the increasing concentrations of the compounds, the constant stimulation mix as in (A) was present.(M and N) Dose response graphs for calcium and cAMP assays using Fluo-4 a.m.(black) and GloSensor (red), respectively, as readouts in PC12 cells treated with increasing concentrations of compound A (M) and compound B (N).In addition to the compounds, the constant stimulation mix was present as in (A).Error bars represent SEM; n = 3 for barcode assays (B-E), Western blots (F-I) and in vitro kinase assays (J, K); n = 6 for luciferase assays, Fluo-4 a.m., and GloSensor assays (L-N).See also FiguresS4 and S5.